RNA interference (RNAi), or RNA silencing, is a novel antiviral mechanism conserved in fungi, plants and invertebrates. RNAi directed viral immunity (RDVI) is a stepwise process that begins with the processing of viral replication intermediates (in the form of double stranded RNA (dsRNA)), into small interfering RNA (siRNA) duplexes by RNase III enzymes. These virus-derived siRNAs (viRNAs) will then be incorporated into an RNA induced silencing complex (RISC) and serve as sequence guide for target viral RNA selection. Within the RISC, an Argonaut (AGO) protein cleaves the viral RNAs selected by viRNAs. Increasing evidence suggests that putative RNA helicases also play important roles in RDVI across kingdoms. However, how these RNA helicases contribute to RDVI remains largely unclear. DRH-1 is a putative RNA helicase conserved in both the Caenorhabditis elegans (C .elegans) nematode and mammals. Previously, our studies have demonstrated that DRH-1 is required for RDVI but becomes dispensable when RNAi is triggered to target cellular transcripts. Because DRH-1 is known to function downstream of siRNA biogenesis, this finding suggests that DRH-1 discriminates invading viral RNAs from cellular transcripts in mediating RNA silencing if we can provide further evidence that siRNAs in cellular gene silencing can mediate efficient virus silencing in the presence of DRH-1. Interestingly, DRH-1 is not conserved in fungi, plants or insects but shares significant sequence identity with RIG-I, a mammalian cytosolic virus sensor. RIG-I discriminates invading viral RNAs from cellular transcripts thereby to induce interferon mediated antiviral innate immunity upon virus detection, our studies thus support a hypothesis that DRH-1 is a RIG-I function analog that discriminates between invading viral RNAs and cellular transcripts thereby to selectively mediate virus silencing irrespective of the origin of silencing siRNAs. To test this hypothesis here we propose to: (1) determine if DRH-1 discriminates replicating viruses from cellular transcripts in mediating RNA silencing regardless of the origin of silencing siRNAs (Specific Aim 1, Research Strategy section, page 4); (2) determine if DRH-1 discriminates between replicating and non-replicating viruses in mediating RNA silencing (Specific Aim 2, Research Strategy section, page 6). The proposed studies are expected to not only help define a functional role for DRH-1 in worm RDVI but also establish C. elegans as a genetic model for mechanistic studies of virus sensing by RIG-I like RNA helicases. Novel findings from the proposed studies may also shed light on some nematode-specific features of RDVI since DRH-1 is not conserved in fungi, plants or insects. PUBLIC HEALTH RELEVANCE: In mammals, including humans, an RNA helicase, termed RIG-I, functions as a virus sensor in antiviral innate immunity. Here we propose to delineate a functional role for DRH-1, a RIG-I homologue found in Caenorhabditis elegans (C. elegans). Because C. elegans represents one of the best genetic models for modern biomedical research the proposed studies have the potential to establish C. elegans as a genetic model for mechanistic study of virus sensing by RIG-I.